Enhancing cancer chemoprevention through rational selection of efficacious combinations of bioactive dietary components is an attractive strategy, due to possible synergistic interactions among these components. However, the use of this approach has been greatly limited by the current poor understanding of dietary component interactions. To explore this promising approach, this project aims to determine the inhibitory effects of two hydroxylated polymethoxyflavones (OH-PMFs, from orange peel) and their combinations on colon carcinogenesis, and also to determine their mode of interaction and the molecular mechanisms involved. The long-term goal is to develop diet-based strategies for colon cancer prevention. Our preliminary results demonstrated that the combination of 5HPMF and 5HHMF (two major OH-PMFs) produced strong synergy not only in inhibiting colon cancer cell growth in culture, but also in inhibiting the formation of aberrant crypt foci (ACF) in azoxymethane (AOM)-treated rats. Based on these results, our central hypothesis is that 5HPMF and 5HHMF act synergistically and produce enhanced inhibitory effects on colon carcinogenesis by synergistically downregulating EGFR and K-Ras. Our rationale is that the successful completion of this project could lead to the development of a novel strategy for colon cancer prevention by using optimized combinations of 5HPMF and 5HHMF from orange peel. The hypothesis will be tested by pursuing two specific aims: 1) Determine the inhibitory actions of 5HPMF, 5HHMF, and their combination in an AOM-induced rat colon carcinogenesis model;and 2) Elucidate the molecular mechanisms of inhibition of colon carcinogenesis by 5HPMF, 5HHMF, and their combination. The innovation in this project is that: 1) it will investigate two novel flavonoid compounds from orange peel, that have not been studied for colon cancer prevention;and 2) The emphasis is on the development of synergistic interactions between the two compounds for their maximized effectiveness, rather than the activities of single isolated compounds. The information obtained from the proposed studies will facilitate the development of novel strategies using 5HPMF and 5HHMF in combination for colon cancer prevention. PUBLIC HEALTH RELEVANCE: The proposed studies focus on potential synergy among bioactive dietary components that is an important and under-investigated area of cancer prevention. Our contribution in this project is expected to be a detailed understanding of the mode of interaction between two unexploited hydroxylated polymethoxyflavones (OH-PMFs, from orange peel) in combination in inhibiting colon carcinogenesis. This contribution is significant because it will provide the knowledge needed to develop a novel colon cancer chemoprevention strategy using combinations of OH-PMFs for their maximized effectiveness, which could have a large positive impact on public health.